Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A

Monika Anand,Bharvi Balar,Rory Ulloque,Stephane R Gross,Terri Goss Kinzy

doi:10.1074/jbc.m601899200

Abstract

Eukaryotic translation elongation factor 3 (eEF3) is a fungal-specific ATPase proposed to catalyze the release of deacylated-tRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperature-sensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.

Highlights

Somes have been reported to possess a compensatory intrinsic ATPase activity, they differ kinetically from the fungal Eukaryotic translation elongation factor 3 (eEF3) [5]
Within the Walker A and B motifs, the nucleotide binding stretch of seven amino acids in ABC1 and -2 are 100% conserved among the ATP-binding proteins [13]
Sites were cloned into a GAL1-10-inducible expression vector 1044, fragments of eEF3 containing these amino acids may with a GST tag at the N terminus (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

GST and His Pulldowns of eEF1A and eEF3—Yeast extracts for in vivo binding assays were prepared by glass bead lysis in TEDG buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 mM DTT, 50 mM KCl, and 1 mM PMSF) from TKY555 with the empty plasmid pTKB544, GSTeEF3 (pTKB705), or the GSTeEF3 fragments (pTKB706, pTKB707, pTKB708, pTKB709, pTKB710). For GST and Ni2ϩ-NTA pulldown assays, 200-␮l reactions containing 50 ␮g of total protein (determined by Bradford reagent; Bio-Rad) and 40 ␮l of either 50% glutathioneSepharose 4B slurry (Sigma) in KETN 150 buffer (150 mM KCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Nonidet, and 1 mM PMSF) or Ni2ϩ-NTA slurry (Amersham Biosciences, GE Healthcare) in buffer C were mixed at 4 °C for 1 h.

RESULTS

A P915L Mutation in an eEF1A

DISCUSSION